Pharmaceutical semi-solid composition of isotretinoin

ABSTRACT

Pharmaceutical semi-solid composition of Isotretinoin An oral pharmaceutical composition of isotretinoin containing at least two lipidic excipients, one of them being hydrophilic (i.e. having an HLB value superior or equal to 10), the other being an oily vehicle

ABSTRACT OF THE DISCLOSURE

The present invention relates to an oral pharmaceutical composition ofisotretinoin containing at least two excipients, one of them beinghydrophilic (i.e. having an HLB value superior or equal to 10), theother being an oily vehicle.

THE PRIOR ART

Isotretinoin (13-cis retinoic acid or 13-cis vitamine A), its isomersand some of its analogs are widely known to have a therapeuticalactivity in the treatment of several severe skin disorders like cysticacne, hypertrophic lupus erythematosus, keratinization disorders. Someevidences have also been brought about the activity of isotretinoin inbasal cell carcinoma and squamous cell carcinoma.

Unfortunately, isotretinoin is also a highly toxic drug. Indeed,although isotretinoin, which is a cis derivative, is known to be lesstoxic than all trans vitamine A derivatives, side effects resulting fromits use such as headache, vomiting, irritation of mucosa and livertoxicity, occur frequently. Furthermore, isotretinoin is known to behighly teratogenic in both animals and humans.

In order to well understand the interest of this invention, it isimportant to briefly summarize the physico-chemical pharmacokineticproperties. Isotretinoin is a reddish-orange powder. It is decomposed inpresence of light and atmospheric oxygen. Isotretinoin is very poorlysoluble in water what mades its bioavailability quite low after an oralintake (25% in fasted conditions and 40% in fed conditions). The maximumconcentration (C_(max)) is reached after 2-4 hours, while the (C_(max))of the active metabolite, 4-oxo-isotretinoin is reached after 6 hours.The elimination half-life of isotretinoin is of 7 to 37 hours while thehalf life (t_(½)) of the active metabolite is of 11 to 50 hours. Thesteady-state concentrations of isotretinoin are reached after 1 week oftreatment.

Very few publications and/or patents about the pharmaceuticalformulation of isotretinoin are available. The drug is available on mostmarkets under the form of a soft gelatine capsule containing a fattyliquid formulation of isotretinoin.

The U.S. Pat. No. 4,464,394 describing for the first time thetherapeutical use of isotretinoin also describes briefly somepossibilities of compositions including it. It involves the use of oneantioxidant agent and of one carrier like lactose, starches orpolyethyleneglycols.

The EP patent 0184942 describes more specific compositions ofisotretinoin involving the use of one antioxidant, one chelating agent,one pharmaceutical carrier and one suspending agent. The compositionobtained is stable during time.

The U.S. Pat. No. 4,545,977 relates to improved compositions ofisotretinoin wherein taurine is associated with isotretinoin to reducethe side effects thereof.

The U.S. Pat. No. 5,716,928 describes a method for increasingbioavailability and for reducing inter and intra individual variabilityof an orally administered hydrophobic pharmaceutical compound, whichcomprises orally administering the pharmaceutical compound with anessential oil or essential oil component in an amount sufficient toprovide greater bioavailability of the active ingredient.

The U.S. Pat. No. 6,028,054 relates to a method for increasingbioavailability of an orally administered hydrophobic pharmaceuticalcompound to human, which comprises orally administering thepharmaceutical compound concurrently with a bioenhancer comprising aninhibitor of e-cytochrome P450 3A enzyme or an inhibitor ofP-glycoprotein mediated membrane transport.

The U.S. Pat. No. 5,993,858 describes a self microemulsifying excipientformulation for increasing the bioavailability of a drug which includesan emulsion including an oil or other lipid material, a surfactant andan hydrophilic co-surfactant.

What is not described is a composition of isotretinoin containing atleast two lipid materials, one of them being hydrophilic. The saidcomposition may be a suspension, emulsion or microemulsion.

BRIEF DESCRIPTION OF THE INVENTION

The advent of high throughput combinatorial chemistry and efficientreceptor based in vitro activity screen has resulted in molecules withpoor physicochemical (ex: dissolution) properties for absorption acrossthe gastro-intestinal tract, like isotretinoin.

It is increasingly being recognized by the pharmaceutical industry thatfor these molecules drug delivery systems play an important role forimproving oral bioavailability.

Although the process of passive diffusion is responsible for absorptionof non ionized lipophilic molecules via the transcellular pathway,specialized absorption mechanisms, first-pass metabolisms and effluxsystems at the gastrointestinal wall appear to play a major role forlack of absorption and poor bioavailability for some molecules.

Isotretinoin is characterized by a low absolute bioavailability and ahigh inter and intra individual variability. Isotretinoin also presentsa wide range of side effects among which some are severe (ocular, skinanemia, hepatic, . . . ). It is consequently of a particular interest todispose of a reliable, stable and highly bioavailable formulation ofisotretinoin.

Several possibilities are available to the formulator to increase thebioavailability of active ingredients (Table A).

TABLE A I. Use of salts, polymorphs. Precursors of the active molecule(=prodrugs) II. Reduction of the particles' size of the active principleand of the excipients used (by trituration, grinding, micronization,precipitation controlled by solvent, temperature or ultrasonics). III.Solid dispersions: Eutectic mixes Solid solutions Vitreous solutions IV.Recrystallization in an aqueous solution of a surfactant V. Modificationof the microenvironment: Hydrophilization pH (acidification) VI.Incorporation of the active principle to lipidic systemsIt has been found that a semi-solid dosage form containing isotretinoinwas advantageous for obtaining a good bioavailability of theisotretinoin. A semi-solid dosage form containing isotretinoin is a formin which isotretoin is mixed with suitable melted excipients. The moltenmix is then filled for example into hard gelatine capsules or otherpharmaceutically acceptable capsules. At ambient temperature(temperature for example of less than 20° C.), the content of thecapsule is solid while at temperature higher than 20° C. (for example attemperature greater or equal to 30° C., advantageously greater or equalto 35° C., preferably substantially at body temperature +/−37° C.), itis liquid or semi-solid (paste). The isotretinoin may be solubilized inthe mix of excipients or partially solubilized. The active ingredientmay also be formulated as a suspension, emulsion or microemulsion.Various lipidic excipients are available to the formulator to obtain asemi-solid formulation. Excipients compatible with hard gelatin capsuleshells are: lipophilic liquid vehicles (refined speciality oils,medium-chain triglycerides and related esters), semi-solid lipophilicvehicles, solubilizing agents, emulsifying agents and absorptionenhancers. The classification of fatty excipients is based on thehydrophilicity or lipophilicity of the excipients, characterized by thehydrophilic/lipophilic balance value (HLB). Examples of lipophilicexcipients are vegetable oils (peanut oil, olive oil, soyabean oil, . .. ), fatty acids (stearic acid, palmitic acid, . . . ), fatty alcohols,. . . Examples of hydrophilic excipients are polyethyleneglycol (PEG)with a molecular weight superior to 3,000. Examples of amphiphilic(=presenting lipophilic and hydrophilic properties) excipients arePoloxamers, Lecithin, PEG esters (Gelucire®), . . . .

The advantages of the semi-solid formulations of the invention aremultiple for isotretinoin: protection of the active ingredient from airand humidity, possibility of increasing the dissolution rate of themolecule and hence of the bioavailability, diminution of the risk ofcontamination of the operator, diminution of the risk of crosscontamination, no possibility of demixing under the effect ofvibrational mixing during manufacturing process, facility of theproduction process. The choice of the nature of the formulation ofcourse influenced the stability of the pharmaceutical form and thebioavailability of the isotretoin contained in it. Generally, a maximumbioavailability is achieved by preparing and keeping the drug in theamorphous/solubilized state in a solid dispersion or in a lipid-basedformulation. For these systems, the barrier we are avoiding is thecompound <<washing-out>> of solution to a large extent into a insolublecrystalline form during the dissolution/release step in vivo.

These systems may consist of suspension, emulsion, microemulsion,self-emulsifying drug delivery systems (SEDDS®) or self-emulsifyingmicroemulsion drug delivery system (SMEDDS®).

Microemulsions have the added advantage over suspensions such asemulsions and dispersions since thermodynamically they are more stable,that they can be manufactured with little energy input and havegenerally a longer shelf-life. Nevertheless, a microemulsion formulationis not a guarantee of higher bioavailability in comparison to suspensiona described hereafter.

The formation of oil-in-water (O/W) and water-in-oil (W/O)microemulsions usually involves a combination of 3-5 basic compoundsi.e. oil, surfactant, cosurfactant, water and electrolytes. Thechallenge is to select for a particular application oil(s) andsurfactant(s) that are acceptable from a toxicological perspective andthat allow to obtain a high bioavailability of the drug, i.e isotretoin.

The assessment of the quality of semi-solid lipid based formulations isquite difficult since the in vitro dissolution test is of little help.Indeed, the in vitro/in vivo correlation between dissolution andbioavailability is very poor for this kind of formulations. Otheranalytical tools are available to the formulator to try to predict thein vivo bioavailability of isotretinoin from various formulations likeCACO-2 cells model, the assessment of the percentage of drug dissolvedin the formulation, differential scanning calorimetry, microscopy, . . ..

Nevertheless, none of them present a guarantee of in vitro/in vivocorrelation and ultimately only pharmacokinetic studies on humansubjects are reliable to assess the bioavailabiltiy of the drug.

DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The pharmaceutical composition of the invention is an oral semi-solidpharmaceutical composition of isotretinoin containing two lipidicexcipients, one of them being hydrophilic i.e. having a HLB value of atleast 10, for example equal to 10, but preferably greater than 10, suchas greater or equal to 12, for example comprised between 12 and 14, andthe other being an oily vehicle.

The pharmaceutical composition of the invention contains advantageouslyat least one hydrophilic excipient with a HLB value of at least 10selected from the group consisting of glyceroyl macrogolglycerides,polyethyleneglycol derivatives, and mixtures thereof. Preferably, thepharmaceutical composition contains from 20 to 80% by weight ofhydrophilic excipient with a HLB value of at least 10 selected from thegroup consisting of glyceroyl macrogolglycerides, polyethyleneglycolderivatives, and mixtures thereof.

The oily vehicle is selected from the group consisting of vegetableoils, medium chain triglycerides, fatty acid esters, amphiphilic oil,glycerol oleate derivative, and mixtures thereof. For example, thecomposition contains from 5 to 70% by weight of an oily vehicle selectedfrom the group consisting of vegetable oils, medium chain triglycerides,fatty acid esters, amphiphilic oil, glycerol oleate derivative, andmixtures thereof.

According to another detail of preferred pharmaceutical compositions ofthe invention, the composition further contains at least one surfactant,preferably selected from the group consisting of sorbitan fatty acidesters, polysorbate derivatives, polyoxyethylene sorbitan fatty acidesters, sodium laurylsulphate, derivatives of lecithine, propyleneglycol esters, fatty acid esters of propylene glycol, fatty acid estersof glycerol, polyethylene glycol, and mixtures thereof. For example, thecomposition contains from 1 to 10% by weight of at least one surfactant.

Furthermore, the pharmaceutical formulation of the invention containsadvantageously at least one disintegrant, preferably selected from thegroup consisting of povidone derivative, sodium croscarmellose andmixtures thereof.

The pharmaceutical composition of the invention may contain one or moresurfactants and/or one or more disintegrants, but contains preferablyone or more compounds acting as surfactants and one or more compoundsacting as disintegrants.

The invention relates also to a pharmaceutical acceptable capsulecontaining at least one semi-solid composition of the invention, forexample at least one composition of the invention as disclosedhereabove. The capsule is for example selected from the group consistingof hard gelatine capsules, soft gelatine capsules, hypromellosecapsules, starch capsules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a ternary diagram of a formulation containing only Gelucire®50/13 and soyabean oil, the third component being water;

FIG. 2 shows the dissolution rate of a reference product(Roaccutane®—20mg active agent), of a suspension containing 20 mgIsotretoin and of an emulsion SEDDS® containing 10 mg Isotretoin;

FIG. 3 shows an In vivo comparative pharmacokinetic profile ofisotretinoin;

FIG. 4 gives the comparative pharmacokinetic profile of differentformulations for isotretinoin,

FIG. 5 gives the comparative pharmacokinetic profile of differentformulations for 4-oxo-isotretinoin, the active metabolite ofisotretinoin and

FIGS. 6 and 7 describe the mean pharmacokinetic profile of isotretinoinand 4-oxoisotretinoin for two formulations.

DESCRIPTION OF EXAMPLES

The present invention relates thus to a semi-solid formulation ofisotretinoin containing at least 2 lipidic excipients, one of them beingan hydrophilic excipient (having a high HLB value namely>10) and theother an oily excipient. The molten mix of these two excipients allowsto totally or partially (depending on the ratio between excipients)dissolve isotretinoin. Different kinds of formulations (SEDDS® orsuspensions) of isotretinoin have been formulated. For suspensions, itwas possible to dissolve a high fraction of isotretinoin in the mix ofexcipients and even the whole quantity of the active ingredient if themanufacturing conditions (high temperature and long time of mixing) andthe formulations were optimized. Excipients particluarly suitable forthe dissolution of isotretinoin were lauroyl Macrogol-32 glycerides(Gelucire® 44/14, Gattefossé) and Stearoyl Macrogol-32 glycerides(Gelucire® 50/13, Gattefossé). When those hydrophilic components aremelted together with an oily vehicle, it allows to obtain very stablesuspensions of isotretinoin in which an important part of the activeingredient is dissolved. A surfactant may also be added to theformulation to still improve the physical stability of the suspension.

SEDDS® formulations of isotretinoin are also stable and may give animproved bioavailability of the drug.

Ternary diagrams allow to observe different areas corresponding todifferent physical states namely coarse emulsion, true emulsion,lamellar solution or micellar solution when the ratio between excipientschanges. The behaviour of the formulation in presence of water changeswhen the ratio changes. One example of this ternary diagram is given inFIG. 1 for a formulation of isotretinoin containing Gelucire® 50/13 andsoyabean oil.

EXAMPLES Example 1 Effect of Different Lipophilic Compounds

The effect of different lipophilic excipients was evaluated in the formof semi-solid capsules. The semi-solid capsules were made by addition ofthe active substance at the pre-melted lipophilic compounds followed bythe filling of the liquid into hard gelatin capsule.

The active substance was incorporated into formulations, listed in tableI, consisting of glyceroyl macrogolglyceride associated with soyabeanoil or derivative, medium chain triglyceride.

TABLE I Formulations n° 1 2 3 4 5 (mg) Isotretinoin 20 20 20 20 20Labrafil ® M1944 CS 132 Gélucire ® 50/02 198 93 Gélucire ® 44/14 217Gélucire ® 50/13 76 60 60 Soya bean oil 304 320 Mygliol ® 320The use of stearoyl macroglyceride (Gélucire® 50/13, Gattefosse) andsoyabean oil allows to obtain a formulation with a dissolution profilesimilar to the reference (Roaccutane® 20 mg, Roche).

The formulation with labrafil or Gelucire® 50/02 are too lipophilic togive a good dissolution in water.

In general, the use of an oily excipient can improve the absorption oflipophilic drug by increasing the solubility of the drug in the lipidicphase, but the release of the active ingredient from the formulation canbe slowed down due to the high affinity of the drug for the oily phase.

The use of dispersed systems (emulsions or suspensions) instead of onlylipophilic or hydrophilic vehicles, improves the absorption of the drugas well as increasing a larger contact surface.

Concerning the Gelucire®, the process of drug release varies accordingto the HLB of the excipient. Gelucire® with high HLB values were foundto be the most favorable for a rapid release of the drug (by diffusionand erosion).

The drug release profiles of the formulations 1 to 5 were evaluated inphosphate buffer pH 7.5 with laurylsulfate and pancreatin. The percentof isotretinoin released after 4 hours is given in the following tableII.

TABLE II percent of isotretinoin released after 4 hours Formulations n°1 2 3 4 5 % released 20.1 69.1 46.0 60.3 78.1The percent of isotretinoin released from the reference (Roaccutane® 20mg) after 4 hours is 55.37%

Example 2 Influence of the Ratio Oily Vehicle/Surfactive Agent on theDissolution and Absorption of the Formulation

The study of the ratio oily vehicle/surfactive agent with theconstruction of a ternary diagram gives information on the dissolutionprofile of the formulation in water.

Stearoyl macrogolglyceride (Gelucire® 50/13) known as a drug solubilizerand emulsifying agent of different drugs (in SMEDDS® or SEDDS®) wastested in association with soyabean oil.

This component has the ability to solubilize a great part ofisotretinoin in the formulation.

This is listed in table III

TABLE III Formulations n° 1 2 3 4 5 6 (mg) Isotretinoin 20 10 10 20 2020 Soyabean oil 270 135 40 152 57 133 Gelucire ® 50/13 84 42 200 228 323247 Filling weight 374 187 260 400 400 400 Ratio oil/Gelucire ® 3.2 3.20.2 0.67 0.17 0.54 50/13

In the presence of water, the behaviour of these formulations aredifferent

-   -   formulations 1 and 2: formation of coarse emulsion with large        droplet sizes    -   formulations 3 and 5: formation of micellar phase or        microemulsion    -   formulations 4 and 6: formation of emulsion with homogeneous        droplet size

The percentage of isotretinoin released increases generally with thepercentage of Gelucire® in the formulation (increased solubility of theactive in this vehicle). For the formulation 1 (ratio oil/Gelucire®50/13=3.2) 54.9% released after 4 hours and for the formulation 3 (ratiooil/Gelucire® 50/13:0.2), 91.2% released after 4 hours.

Dissolution Test

For poorly soluble molecules, the prediction power of the in vitrodissolution test is weak since the in vitro/in vivo correlation is knownto be poor. Nevertheless, an optimized dissolution test (using enzymesand surfactant) is of some help to assess the rate of release of thedrug from the lipidic composition. It must be noted that the conditionsof the dissolution (dissolution medium, speed of the paddles,temperature, . . . ) test influence dramatically the results of the testand should consequently be standardized to allow comparison betweenvarious formulations.

The conditions of the solution test used for assessing the dissolutionof isotretinoin were the following:

-   -   paddle apparatus    -   150 rpm    -   37° C.    -   buffer pH 7.5 with laurylsulfate 2.5% and pancreatin 1 g/L

FIG. 2 shows the dissolution rate of a reference product (Roaccutane®—20mg active agent), of a suspension containing 20 mg Isotretoin and ofan emulsion SEDDS® containing 10 mg Isotretoin (formulation givenhereinbelow).

As the information brought by the dissolution test is poor in term ofcorrelation with in vivo bioavailability, it is of interest to disposeof other means to predict the in vivo bioavailability.

The caco-2 cell culture system can be used for determining permeabilityof compounds (especially for poorly soluble compounds). The caco-2 cellmodel allows to measure the transport of drug from the apical to theserosal side as well as from the serosal to the apical side. This allowsto determine if an efflux system is operational.

The caco-2 cells model is interesting because:

-   -   The cells used are from human origin (contrary to the models        using segments of animal's guts). They are coming from an        adenocarcinoma of the human colon but spontaneously        differentiate into small intestine's epithelail cells. When put        in culture, they form a monolayer of polarized cells expressing        several enzymatic systems.    -   It offers a better prediction of the human intestine absorption        than the animals models    -   The reproducibility of the test is relatively high    -   It allows to take samples from both apical and basolateral sides

Caco-2 cells experiments have been performed with one SEDDS® and onesuspension isotretinoin formulations.

RESULTS

It was first proven that neither the active ingredient nor the excipientused in the formulations were toxic for the cells. It was also proventhat the integrity of the membranes of the cells was maintained duringthe whole experience.

Methodology:

The formulations tested are put in solution in 250 ml of BME.Taurocholate (10 mM) was added to the solutions to better mimate the inphysiological conditions. The different solutions so prepared are put incontact with Caco-2 cells at the apical or basolateral side. The cellsculture inserts) are incubated for 3 hours at 37° C. and samples of 100μl are taken every hours

The formulations tested were the following:

-   -   Formulation SEDDS® (batch number 26F97/1):    -   Isotretinoin: 10 mg    -   Gelucire® 50/13: 134 mg

Phospholipon 90®: 11 mg

-   -   Tween 80®: 71 mg    -   IPP®: 24 mg    -   Pro capsula una

Formulation suspension (batch number 25F97/1)

-   -   Isotretinoin: 20 mg    -   Gelucire® 50/13: 83.7 mg    -   Soyabean oil: 270 mg    -   Procapsula una

RESULTS

Passage of formulations from apical side→basolateral side

Time SEDDS ® Suspension SEDDS ® + Suspension + minutes (26F97/1)(25F97/1) TC TC control 60 0.7721 0.6708 0.7019 0.6469 0.0718 120 2.40960.8749 1.4347 0.9513 0.1836 180 2.6226 1.1311 3.2419 1.5073 0.6156Passage of formulations from basolateral side apical side

Time SEDDS ® Suspension SEDDS ® + Suspension + minute (26F97/1)(25F97/1) TC TC control 60 2.0496 0.3948 8.1291 0.8713 0.0650 120 3.08440.9068 8.3496 1.8460 0.1131 180 4.3653 1.0763 9.7110 2.0779 0.1481The results demonstrate that the passage of isotretinoin is superior forthe SEDDS® formulation than for the suspension formulation. In order toconfirm these results, a comparative pharmacokinetics study has beenperformed.

PK Studies

The bioavailability of SEDDS® (26F97/1) and suspension (25F97/11)isotretinoin formulations has been assessed and compared to thebioavailability of the reference (Roaccutane® 20 mg, Hoffman LaRoche) onsix healthy volunteers in a single dose, three way, cross-overpharmacokinetic study). The drug was taken with food (standardizedbreakfast). The plasma concentration of isotretinoin and its activemetabolite 4-oxo-isotretinoin were quantified using a fully validatedLC/MS/MS method.

The FIG. 3 described the mean pharmacokinetic profile obtained for eachformulation.

The following table gives the value of the main pharmacokineticsparameters obtained for each formulation of isotretinoin.

AUC_(72 h) C_(max) T_(max) Formulations (ng · h/ml) (ng/ml) (h)Roaccutane ® 20 mg 1747.89 116.63 1.83 (96C15315AA) Suspension 20 mg4308.72 230.96 5.67 (25F97/1) SEDDS ® 10 mg 1494.64 98.36 3.00 (26F97/1)It appears that both the SEDDS® and the suspension formulation are ableto significantly increase the bioavailability of isotretinoin incomparison to the marketed reference. Indeed the ratio between AUC_(72h)of the supension 20 mg and Roaccutane® 20 mg is of 2.47. The SEDDS® 10mg present an AUC_(72h) similar to this of Roaccutane® 20 mg what meansan approximately 2-fold increase of bioavailability (ratio AUC_(72h)SEDDS® 10 mg/AUC_(72h) Roaccutane® 20 mg=0.86). Furthermore, thesuspension and SEDDS® formulations both presented a lowerintraindividual variability of the bioavailability as demonstrated bythe values of relative standard deviations (rsd) which are of 36.0%,22.72% and 28.18% for Roaccutane® 20 mg, suspension 20 mg and SEDDS® 10mg respectively.

Nevertheless, the results obtained in vivo are not correlated with theresults obtained on caco-2 cells since on this model the permeability ofthe SEDDS® formulation was much higher than the permeability of thesuspension formulation while in vivo the suspension formulation givesthe best results.

A second pharmacokinetic study was performed on completely differentformulations (6 subjects, 2-way, fed, cross-over study). Those wereformulations of isotretinoin under the form of a suspension in which theratio between Gelucire® 50/13 and soyabean oil was very different thatthe previous formulation of suspension

The two formulations tested were the following:

-   -   F1: suspension without surfactant (batch number H23K99/1)    -   Isotretinoin: 20 mg    -   Gelucire® 50/13: 247 mg    -   Soyabean oil: 133 mg    -   F2: suspension with surfactant (batch number H07L99/1)    -   Isotretinoin: 20 mg    -   Gelucire® 50/13: 240 mg    -   Soyabean oil: 130 mg    -   Span 80®: 20 mg

The FIG. 4 gives the comparative pharmacokinetic profile of eachformulation for isotretinoin

The FIG. 5 gives the comparative pharmacokinetic profile of eachformulation for 4-oxo-isotretinoin, the active metabolite ofisotretinoin.

In order to confirm the first bioavailability data obtained with thepresent invention, a larger pharmacokinetic study has been performed.

The bioavailability of a capsule of isotretinoin 16 mg (see theformulation herebelow) from the present invention has been assessed andcompared to the bioavailability of the reference (ROACCUTANE® 20 mgcapsule, Roche) on 24 healthy subjects.

This study (SMB-ISO-SD011) was a single dose, two treatment, two period,two sequence, randomised, crossover and with at least 18 days wash-outbetween the two periods.

The subjects were healthy caucasian volunteers of both sexes(non-pregnant, non-breast-feeding), aged 18 to 50 years, non smokers orsmoking less than 10 cigarettes per day.

The drugs was taken with food (a standardized breakfast).

Blood samples were collected according to the following samplingschedule: pre-dose and 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 10 h, 12h, 14 h, 24 h, 36 h, 48 h, 72 h, 96 h, 120 h, 168 h and 216 hourspost-dose.

The plasma concentration of isotretinoin and its active metabolite4-oxo-isotretinoin were quantified using a fully validated LC/MS/MSmethod. The continuous variables were evaluated according to anunivariate ANOVA, based on log-transformed data. The Wilcoxonnon-parametric ANOVA were used where appropriate. Bioequivalence wasevaluated using the Shuirman two one-sided t-test (90% Cl) and thewestlake single sided confidence interval (95% CL)

The FIGS. 6 and 7 describe the mean pharmacokinetic profile ofisotretinoin and 4-oxoisotretinoin for the two formulations (n=24subjects) while the tables herebelow give the comparative mainpharmacokinetic parameters.

Formulation of isotretinoin 16 mg (mg/capsule)

isotretinoin 16 stearoyl macrogol glycerides (Gelurire 50/13 ®) 192 soyabean oil refined 104 sorbitane oleate (Span 80 ®) 16As seen, the dose of 16 mg of the formulation corresponding to thepresent invention gives a bioavailability similar to 20 mg of themarketed formulation, what is the evidence of the supra-bioavailabilityof the formulation corresponding to the present invention.

The tables hereinbelow gives the value of the main pharmacokineticsresults and statistical analysis obtained for each formulation ofisotretinoin and 4-oxoisotretinoin.

This study demonstrated that ROACCUTANE® 20 mg and isotretinoin 16 mg xare bioequivalent after a single oral dose administration of eachproduct in fed conditions. Indeed, the primary parameters AUC (AUC_(∞)and AUC_(216 h)) were within the predetermined confidence interval.

This study demonstrated also that Isotretinoin 16 mg has a safetyprofile comparable with that described in the literature for otherisotretinoin preparations and similar to this of ROACCUTANE® 20 mg.

Pharmacokinetic results and statistical analysis of comparative study in24 volunteers for isotretinoin (log-transformed data)

Bioequivalence tests Results Shuirman Westlake Parameter ROACCUTANE ® 20mg Isotretinoin 16 mg 90% CI Range 95% CL AUC_(∞) 5657.09 (ng · h/ml)5696.92 (ng · h/ml) 92-123 19.07 ± SD ± 2682.98 ± 1938.89 ± RSD ± 47.42± 34.03 AUC_(216 h) 5601.36 (ng · h/ml) 5664.39 (ng · h/ml) 92-124 19.51± SD ± 2670.85 ± 1953.52 ± RSD ± 47.68 ± 34.48 C_(max) 386.68 (ng/ml)441.79 (ng/ml) 103-140  28.81 ± SD ± 218.21 ± 197.43 ± RSD ± 56.43 ±44.68 T_(max) 4.92 (h) 4.50 (h) / / ± SD ± 2.22 ± 0.66 ± RSD ± 45.24 ±14.65

Pharmacokinetic results and statistical analysis of comparative study in24 volunteers for 4-oxoisotretinoin (log-transformed data)

Bioequivalence tests Results Shuirman Westlake Parameter ROACCUTANE ® 20mg Isotretinoin 16 mg 90% CI Range 95% CL AUC_(∞) 5750.36 (ng · h/ml)5769.04 (ng · h/ml) 92-124 19.65 ± SD ± 2717.38 ± 2161.97 ± RSD ± 47.26± 37.48 AUC_(216 h) 5638.32 (ng · h/ml) 5712.21 (ng · h/ml) 92-125 20.46± SD ± 2704.73 ± 2126.61 ± RSD ± 47.80 ± 37.23 C_(max) 111.52 (ng/ml)115.15 (ng/ml) 94-125 20.18 ± SD ± 69.62 ± 66.25 ± RSD ± 62.43 ± 57.53T_(max) 17.83 (h) 16.33 (h) / / ± SD ± 10.60 ± 10.11 ± RSD ± 59.43 ±61.88

1-13. (canceled)
 14. An oral pharmaceutical composition in the form of acapsule comprising a semisolid suspension or a solution of isotretinoinand at least a first and a second lipidic excipient, the compositionexhibiting a dissolution of at least 40% within 4 hours as measuredusing USP paddle apparatus at 150 rpm in an aqueous dissolution mediumconstituted by buffer of pH 7.5, sodium lauryl sulfate 2.5% andpancreatin 1 g/L at 37° C.
 15. The oral pharmaceutical composition ofclaim 14, wherein the composition exhibits an isotretinoin release rateof at least 60% within 4 hours.
 16. The oral pharmaceutical compositionof claim 14, wherein isotretinoin comprises between 1% to 25% w/w of thecomposition, the first lipidic excipient comprises between 20% to 80%w/w of the composition and the second lipidic excipient comprisesbetween 5% to 70% w/w of the composition.
 17. The oral pharmaceuticalcomposition of claim 14, wherein the first lipidic excipient is selectedfrom the group consisting of glyceroyl macrogolglycerides, polyethyleneglycol esters, and mixtures thereof.
 18. The oral pharmaceuticalcomposition of claim 14, wherein the second lipidic excipient isselected from the group consisting of vegetable oils, medium chaintriglycerides, fatty acid esters, glycerol oleate and mixtures thereof.19. The oral pharmaceutical composition of claim 14, wherein the firstlipidic excipient has a HLB value of at least
 12. 20. The oralpharmaceutical composition of claim 14, wherein the first lipidicexcipient has a HLB value of at least
 13. 21. The oral pharmaceuticalcomposition of claim 14, wherein the composition is a semi-solidsuspension.
 22. The oral pharmaceutical composition of claim 14, whichfurther comprises at least one surfactant.
 23. The oral pharmaceuticalcomposition of claim 22, wherein the surfactant is selected from thegroup consisting of sorbitan fatty acid esters, polysorbate compounds,polyoxyethylene sorbitan fatty acids esters, sodium lauryl sulphate,compounds of lecithin, propylene glycol esters, fatty acid esters ofpropylene glycol, fatty acid esters of glycerol, mixtures thereof. 24.The oral pharmaceutical composition of claim 22, wherein the surfactantcomprises between 1% to 10% w/w of the composition.
 25. The oralpharmaceutical composition of claim 14, which further comprises at leastone disintegrant.
 26. The oral pharmaceutical composition of claim 25,wherein the disintegrant is selected from the group consisting ofcrospovidone, sodium croscarmellose, and mixtures thereof.